Process for the long-term surviving culture of hepatocytes

Abstract

A process is disclosed for the long term surviving culture of hepatocytes maintaining their morphological and functional characteristics for long-term periods, suitable to assest the acute and chronic hepatotoxicity of drugs, chemicals and environmental pollutants. The long term surviving culture of hepatocytes is carried out in a culture media containing fibroblast cells or fibroblast cell products treated to prevent cell multiplication and inoculated under controlled density.

What is claimed is: 
     
       1. A process for the long-term surviving culture of hepatocytes, comprising: culturing said hepatocytes at a density of from about 180 to about 65,000 cells/cm 2  in a culture medium containing from about 1 to about 100 ug/ml of hydrocortisone, in the presence of either fibroblast cells treated to prevent their multiplication and to maintain normal morphology and expression of hepatocyte function for long-term periods, or fibroblast cells products on a modified substrate, at controlled densities of from about 30,000 to about 65,000 cells/cm 2 . 
     
     
       2. The process as claimed in claim 1, wherein said fibroblast cells are 3T3 fibroblast cell layers treated to prevent their multiplication and the overgrowth of the hepatocytes. 
     
     
       3. The process as claimed in claim 2, wherein said 3T3 fibroblast cell layers are treated with an agent which damages DNA to prevent their multiplication and the overgrowth of hepatocytes. 
     
     
       4. The process as claimed in claim 3, whrein said agent which damages fibroblasts DNA is mitomycin-C. 
     
     
       5. The process as claimed in claim 2, wherein said 3T3 fibroblast cell layers are treated with a ionizing radiation to prevent their multiplication. 
     
     
       6. The process as claimed in claim 5, wherein said inoizing radiation is carried out with x or gamma rays. 
     
     
       7. The process as claimed in claim 1, wherein said culture medium contains the Dulbecco modification of Eagle medium. 
     
     
       8. The process as claimed in claim 1, wherein said culture medium contains the Dulbecco modification of Eagle medium suplemented with about 10% calf serum. 
     
     
       9. The process as claimed in claim 1, wherein said culture medium also contains hormones. 
     
     
       10. The process as claimed in claim 1, wherein said culture medium contains vitamines and growth factors. 
     
     
       11. The process as claimed in claim 1, wherein said culture medium also contains insulin. 
     
     
       12. The process as claimed in claim 1, wherein said culture contains about 5 to 10 μg/ml of hydrocortisone to inhibit viable fibroblast cells present in the hepatocytes. 
     
     
       13. The process as claimed in claim 1, wherein said culture medium also contains modifiers of mixed-function oxidases. 
     
     
       14. The process as claimed in claim 13, wherein said modifiers are selected from the group consisting of about 2×10 -3  M phenobarbital, 3-methylcholanthrene, Aroclor 1254 and SKF 525-A. 
     
     
       15. The process as claimed in claim 1, wherein said culture medium also contains a medium conditioned by the fibroblasts 
     
     
       16. The process as claimed in claim 1, wherein 2×15 -3  M phenobarbital is added to said hepatocytes to exert an induction of Cytochrome P-450 activity increasing its content up to 2-fold. 
     
     
       17. The process as claimed in claim 1 wherein said 3T3 fibroblast cells are inoculated at a density from about 30,000 to about 65,000 cells/cm 2  and said hepatocytes at a density from about 180 to about 35,000 cells/cm 2 . 
     
     
       18. The process as claimed in claim 1, wherein said hepatocytes are obtained from humans, mammals, birds, reptiles, amphibians and fishes. 
     
     
       19. A long-term surviving hepatocyte culture, comprising: hepatocyte cells at a density of from about 180 to about 65,000 cells/cm 2  in a culture medium containing from about 1 to about 100 μg/ml of hydrocortisone, in the presence of either multiplication inhibited fibroblast cells or fibroblast cell products on a modified substrate, at controlled densities of from about 30,000 to about 65,000 cells/cm 2 , to maintain their long-term survival characteristics. 
     
     
       20. A long-term surviving hepatocyte culture as claimed in claim 19, wherein the culture medium contains a medium conditioned by the fibroblast cells. 
     
     
       21. The long-term surviving hepatocyte culture as claimed in claim 19, wherein said fibroblast cells are multiplication inhibited 3T3 fibroblast cell layers. 
     
     
       22. The long-term surviving hepatocyte culture as claimed in claim 19, wherein said culture medium also contains the Dulbecco modification of Eagle medium. 
     
     
       23. The long-term surviving hepatocyte culture as claimed in claim 19, wherein said culture medium also contains the Dulbecco modification of Eagle medium suplemented with about 10% calf serum. 
     
     
       24. The long-term surviving hepatocyte culture as claimed in claim 19, wherein said culture medium also contains hormones. 
     
     
       25. The long-term surviving hepatocyte culture as claimed in claim 19, wherein said culture medium also contains vitamines and growth factors. 
     
     
       26. The long-term surviving culture of hepatocytes as claimed in claim 19, wherein said culture medium also contains insulin. 
     
     
       27. The long-term surviving hepatocyte culture, as claimed in claim 19, wherein said culture medium also contains modifiers of mixed-function oxidases. 
     
     
       28. The long-term surviving hepatocyte culture as claimed in claim 27, wherein said modifiers are selected from the group consisting of about 2×10 -3  M phenobarbital, 3-methylcholantrene, Aroclor 1254 and SKF 525-A. 
     
     
       29. The long-term surviving culture of hepatocytes as claimed in claim 19, wherein said culture medium contains 2×10 -3  M phenobarbital. 
     
     
       30. The long-term surviving hepatocyte culture as claimed in claim 19, wherein said hepatocytes are human, mammals, birds, reptiles, amphibian or fish hepatocytes.